Non antibiotic selectable markers for live vaccines

ABSTRACT

The invention relates to DNA constructs encoding a safe, selectable marker, other than an antibiotic resistance marker, vectors and/or cells including said constructs; and vaccines based on said constructs for use in animals and particularly humans. The safe, selectable marker being a marker that confers resistance to an agent, other than an antibiotic, which would otherwise deleteriously affect the growth of a cell in which said construct was placed.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of International Application PCT/GB97/01080, withan international filing date of Apr. 18, 1997, now abandoned.

FIELD OF THE INVENTION

The invention relates to DNA constructs encoding a selectable markerother than an antibiotic resistance marker; vectors and/or cellsincluding said constructs; and vaccines based on said constructs for usein animals and particularly, but not exclusively, for use in humans.

As referred to above antibiotic means any of various chemical substancessuch as penicillin, ampicillin, streptomycin, neomycin or tetracyclineproduced by various microorganisms, or their synthetic counterparts.

Description of the Related Art

There is an ongoing need to immunise animals, and in particular humans,against various viruses, bacteria and parasites. The aim of immunisationis typically to elicit a secretory, humoral or cell-mediated immuneresponse to at least one antigen expressed by said virus, bacteria orparasites. To this end, a number of vaccines have been developed, someinvolving the administration of live oral strains of bacteria such asthe live oral salmonella vaccines which are typically based upon strainsof salmonella which have been attenuated by the introduction of anon-reverting mutation in a gene(s) in the aromatic biosyntheticpathway, or a stress protein such as HtrA, of the bacteria. Since theseoral vaccines were first developed they have been used as carriers fordelivering other selected immunogenic antigens and thus serving as whatis termed multivalent vaccines i.e. they confer resistance against morethan one diseased state. For example, it is known that an attenuatedstrain of Salmonella typhi (Ty21A), which is currently administrated asan oral vaccine against typhoid, can be engineered, typically bychromosomal integration, so as to genetically express at least oneantigen from at least one other pathogen. This sort of geneticmanipulation does not require the use of a selectable marker. Thetechnique is extremely desirable because the single administration ofone vaccine can provide protection against a multitude of diseases.

However, unfortunately, the above multivalent vaccines expressrecombinant antigen using a single gene copy of the relevant antigen andthus the level of expression is low i.e. less than 1% of total cellprotein. For example the expression level of a single copy malarialantigen gene from the chromosome of Salmonella typhi has been estimatedto be 0.16% of total cell protein (Gonzalez et al 1994). From 10 humanvolunteers who were vaccinated, in only 3 volunteers were there actuallyany detectable immune responses (Gonzalez et al 1994). In addition suchvaccines may provide for an immune response that is less than desirable,typically in a healthy individual the course of the disease appearsunaffected and quantifiable in vitro tests of a routine nature for thesame individual are comparably poor.

Thus, contrary to expectations, a multivalent vaccine may in practiceonly be effective against a single pathogen.

Theoretically, it should be possible to improve the immunogencity of therecombinant antigen by increasing its level of expression. This could bedone either by providing for multiple copies of the gene encoding therelevant antigen(s) and/or by genetically engineering the gene so thatit is linked to an agent, such as a promoter, providing for highexpression. However, in order to assess the success of such geneticengineering it is necessary to include in the engineering processselectable markers so that successfully transformed cells, suitable foruse as vaccines, can be identified. Markers that are currently used areantibiotic resistant markers and the tests for successful transformationtypically involve exposure of a cell population to a selected antibioticand subsequent isolation of those cells showing antibiotic resistance.

This extensively used test for determining successful transformationcannot be employed in the manufacture of vaccines for use in eitherhumans or animals. This is because incorporating a gene conferringantibiotic resistance into a vaccine could be potentially hazardous fora number of reasons. Firstly, if an adverse reaction occurred in anunusually suspectable vaccine, the antibiotic could not be administeredto the individual to clear the vaccine strain. Secondly it is of concernthat the antibiotic resistance gene could be transferred to othermicroorganisms rendering them no longer amenable to control with theantibiotic.

Thus it can be seen there is a need to provide for multivalent vaccinesand in order to achieve this it is necessary to ensure that recombinantgenes encoding pre-selected antigens are expressed to a sufficient levelto elicit an immune response. Thus there is a need to use vectors thateither comprise multiple copies of the relevant gene and/or at least onecopy of the relevant gene operatively linked to a high expression systemsuch as a high expression promoter when transforming a host cell such asan attenuated strain of Salmonella typhi. However, to do this,transformation must be assessed using a selectable marker and there istherefore a need to identify a selectable marker that can be used totransform a host cell which, ultimately, will be administered to, orotherwise harboured within, an animal and in particular a human. Thusthere is a need to find a safe selectable marker and by this term wemean a marker that can be used in a system, or a cell that willeventually be given to an animal and in particular humans.

DESCRIPTION OF THE INVENTION

It is therefore an object of the invention to provide a multivalentvaccine which is suitable for administration to animals, andparticularly but not exclusively, humans.

It is yet a further object of the invention to provide a safe,selectable marker for use, in, or in relation to animal systems.

It is yet a further object of the invention to provide a vectorincluding said safe, selectable marker.

It is yet a further object of the invention to provide a vectorincluding said safe, selectable marker and also at least one geneencoding a selected recombinant antigen which is preferably, but notexclusively, operatively coupled to a high expression promoter.

We have achieved the objects of the invention by identifying a gene thatencodes for an agent, other than an antibiotic resistance conferringagent, which is able to counter, or advantageously affect, the otherwisedeleterious effect of a given selected substance on a cell to betransformed by a vector including said gene.

In one embodiment of the invention the invention is achieved by use ofthe BIALAPHOS resistance gene (bar gene).

The gene conferring resistance to BIALAPHOS (the bar gene) was clonedfrom Streptomyces hygroscopicus genomic DNA in 1986 by Murakmi et al.

The bar gene encodes for phosphinothricin acetyltransferase (PAT) whichconverts the herbicide DL-phosphinothricin (PPT) [CAS No. 77182-82-2Bellinger R. R., et al Weed Science 33:779 1985], with high affinity,into a non herbicidal acetylated form by transferring the acetyl groupfrom acetyl CoA onto the free amino group of PPT. The reaction mechanismis illustrated in FIG. 1 (Thompson et al).

In the absence of the bar gene PPT is an analogue of glutamate and aspecific and very strong inhibitor of glutamine synthetase in bothplants and bacteria. Glutamine synthetase plays a central role in theassimilation of ammonia and the regulation of nitrogen metabolism. Infact it is the enzyme that detoxifies ammonia. Thus, in plants, when PPTis applied ammonia metabolism is disturbed and ammonia accumulates totoxic levels in the cells. In bacteria, PPT acts as a bacteriostaticagent as a result of glutamine starvation, in a media lacking this aminoacid, as it irreversibly inhibits glutamine synthetase (D'Halluin et al1992).

As a result of the above described activity of the bar gene product itis possible to use the bar gene as a safe, selectable marker in geneticengineering experiments where it would be otherwise hazardous to usegenes encoding substances which can be used by pathogens to obtainresistance to therapeutic agents which are related to said substances,such as antibiotic resistant genes.

In its broadest aspect the invention therefore concerns the use of asafe, selectable marker, i.e. a gene that confers resistance to an agentother than an antibiotic, which agent can be used to deleteriouslyaffect the growth of an organism transformed so as to include at leastsaid marker.

According to a first aspect of the invention there is therefore provideda transformed cell which has been engineered so as to express at leastone antigen, homologous or heterologous, and at least one safe,selectable marker which confers on said cell resistance to an agent,other than an antibiotic, which would otherwise deleteriously affect thegrowth of said cell.

Ideally, expression of said antigen is of a sufficient level to elicitan immune response when said transformed cell is administered to orgiven to an animal. Thus, multiple copies of the gene encoding saidantigen may be provided in said transformed cell and/or at least onecopy of said gene is operatively linked to a high expression agent suchas a high expression promoter.

In one aspect the invention may involve the genetic manipulation of abacterial cell so that it expresses at least one antigen, i.e. it isunivalent, and in this instance ideally multiple copies of the geneencoding said antigen will be engineered into said cell along with asafe, selectable marker so that successful transformation can bemonitored.

Alternatively, said at least one antigen may include a number ofdifferent antigens so as to confer on said cell multivalency, and onceagain, preferably a plurality of copies of the genes encoding therelevant antigens may be provided so as to enhance expression of saidantigens. Alternatively or in addition at least one of said antigens islinked to a high expression promoter, and preferably said multiplecopies of said antigen; or preferably multiple copies of said multipleantigens maybe linked to a high expression promoter.

Ideally, said promoter is inducible, and preferably inducible in vivo.For example one such promoter is the E. coli nitrite reductase promoter,or indeed any other promoter which would favour a high expression of agene coupled thereto, especially in in vivo conditions.

In any of the above instances successful transformation of said cell ismonitored by the safe, selectable marker.

In a preferred embodiment of the invention said cell is of the strainSalmonella such as, for example, Salmonella typhi. In this instance thecell is ideally an attenuated strain of Salmonella typhi. In addition,in this instance, this cell expresses antigens relating to typhoid feverand the cell is either transformed, less preferably, to express more ofsaid antigens conferring resistance to typhoid fever, or morepreferably, to express antigens of a different pathogenic type so as toconfer resistance to other pathogens (Khan et al 1994; Chabalgoity et al1996).

In yet a further preferred embodiment of the invention the said cellincludes a construct expressing at least a fragment of tetanus toxoid,and ideally expressing the highly immunogenic but atoxic fragment C(TetC) from tetanus toxin. (Khan et al 1994). However, it is within thescope of the invention for said cell to include any other preferredconstruct which enhances the immunogenicity of the cell and thusincreases the desirability of the use of the cell as a vaccine. One suchfurther example would be the B sub unit of Vibrio cholerae or the B subunit of Escherichia coli.

It will be apparent from the above that where said cell, prior totransformation, expresses an antigen, whether homologous or heterologousand thus is univalent, subsequent to transformation, assuming aheterologous antigen is used, then said univalent cell will becomemultivalent. Moreover, it is possible to increase the valency of thecell by the process of transformation. For example, where said cellexpresses either at least one homologous antigen and at least oneheterologous antigen, or at least two heterologous antigens, thenfollowing the process of transformation, assuming transformation withyet a further heterologous antigen, the valency of the cell will beincreased so as to potentially confer resistance against a number ofpathogens equal to or less than number of antigen types.

According to a yet further aspect of the invention there is provided avector comprising at least one gene encoding an agent, other than anantibiotic resistance agent, that counters, or advantageously affects,the otherwise deleterious effects of a substance to which a cell that isto be transformed by said vector is susceptible; and at least one geneencoding a pre-selected antigen.

According to yet a further aspect of the invention there is provided avector comprising at least one bar gene and at least one gene encoding apre-selected antigen from at least one pathogen known to cause diseasein animals.

Preferably either vector includes multiple copies of said gene encodingsaid antigen and/or copies of different genes encoding differentantigens all selected from pathogens which are capable of causingdisease in animals.

More preferably still at least one of the aforementioned genes isoperatively coupled to a high expression promoter and ideally at leastone of said antigens is coupled to said promoter so as to provide forhigh expression of at least one of said antigens.

According to a yet further aspect of the invention there is provided avector including an antibiotic resistance gene into which gene has beeninserted a gene encoding resistance to a substance, other than anantibiotic, which substance is capable of deleteriously affecting a cellto be transformed by said vector.

In this aspect of the invention the said antibiotic resistance gene isrendered insertionally inactivated.

In a preferred embodiment of this aspect of the invention the said geneencoding resistance to the substance is the bar gene.

According to yet a further aspect of the invention there is provided avector including an antibiotic resistance gene into which there has beeninserted a gene encoding resistance to a substance, other than anantibiotic, which deleteriously affects the growth of a cell into whichsaid vector is to be inserted; and also at least one gene encoding aselected antigen.

In a preferred embodiment of the invention said selected antigen isheterologous having regard to the nature of the cell to be transformedby said vector.

More preferably further still said vector comprises multiple copies ofsaid antigen and/or at least one, and preferably multiple copies, of atleast one other antigen of at least one other pathogen.

Preferably further still at least one of said genes is operativelycoupled to a high expression promoter, and ideally, at least one of saidgenes encoding at least one of said antigens is operatively coupled tosaid high expression promoter.

According to a yet further aspect of the invention there is provided DNAconstructs encoding the characterising parts of the above referred tovectors.

According to a yet further aspect of the invention there is provided avaccine for use in animals comprising the aforementioned cell and/orvector of the invention.

Suitable antigens for working the invention include, but are not limitedto, antigens relating to human immuno-deficiency virus (HIV) such asHIV-1 or HIV-2; the CD4 receptor binding site for HIV; hepatitis A or Bvirus; human rhinovirus such as type 1 or type 14; Herpes simplex virus;poliovirus type 2 or 3; foot-and-mouth disease virus; rabies virus;rotavirus; influenza virus; coxsackie virus; human papilloma virus suchas type 16, the E7 protein thereof, and fragments containing the E7protein; simian immunodeficiency virus; antigens from Bordetellapertussis such as the P69 protein and FHA antigens; Vibrio cholerae;Bacillus anthracis; and E.coli antigens such as LT-B antigens, K88antigens and enterotoxigenic antigens. Other antigens include the CD4antigen, Schistosome mansoni antigens such as P28 antigens, antigens offlukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, andmalaria parasites for example parasites of the genus Plasmodium orBabesia. Other antigens include those derived from the mycobacteria.

As mentioned, suitable promoters for use in the invention includepromoters which are ideally inducible and so respond to a change in theenvironment.

An example is a promoter that is inducible having regard to anaerobicconditions such as the nirB promoter.

Suitable cells for working the invention comprise attenuated bacteriasuch as those selected from the genus Salmonella, Haemophilus,Neisseria, Bordetella, Vibrio or Yersinia, or attenuated mycobacteria.Details of attenuated bacteria are well know to those skilled in the artand will not be described in detail hereinafter.

The vaccine of the invention may comprise at least one suitable adjuvantideally, the vaccine is provided in a suitable form for oraladministration for example in a capsular form in which the vaccine islyophilised. Alternatively, the lyophilised vaccine may be provided inthe form of a suspension suitable for reconstitution prior toadministration. Typically reconstitution is provided using suitablebuffer to ensure the viability of the organisms. Where the vaccine is tobe orally administrated it is desirable to pre-administer an alkalinepreparation such as sodium bicarbonate in order to safeguard against theeffects of gastric acidity. Alternatively still the vaccine may besupplied in the form of an aerosol.

It will be apparent to those skilled in the art that the dose of thevaccine will be dependant upon a number of variables, not least, thesize and weight of the vaccine recipient, the type of vaccine formulatedand the immunogenicity of the relevant antigen(s).

An embodiment of the invention will now be described by way of exampleonly with reference to the following figures wherein:

FIG. 1 shows the reaction mechanism catalysed by the bar gene productPAT;

In the following experiments a gene encoding resistance to a substanceother than an antibiotic is described and in particular the bar geneencoding resistance to the herbicide PPT. However, it is not intendedthat the application should be limited by this example rather thisexample is provided by way of comprehension only.

Salmonella tvphimurium Experiments to Show that the Selected Host Cellis Sensitive to PPT

Strains of E.coli (TG2) and S. typhimurium (CS) were incubated overnightat 37° C. under normal culture conditions and in normal culture medium.Cells from the overnight culture were diluted into phosphate bufferedsaline (PBS) and a series of serial dilutions plated onto LB plates withPPT at concentrations of 250 ug/ml and 0 ug/ml.

As a result of this incubation we established that in the absence of PPTgrowth of the two strains proceeded as normal. However growth wastotally inhibited at PPT concentrations of 250 ug/ml at dilutionsranging from 10¹ and 10⁵ of the overnight culture.

This experiment therefore established that our selected host cell wassuspectable to PPT.

Experiments to Replace the Antibiotic Resistance Marker of a SalmonellaExpression Plasmid with the Bar Gene

The plasmid pTETnir15 expresses from the nirB promoter fragment C (TetC)of tetanus toxin (Chatfield et al 1992).

This plasmid contains the gene encoding for beta-lactamase (ampR) whichconfers resistance to the antibiotic ampicillin. Hence ampicillinresistance is the selectable marker. Ideally we need to introduce thebar gene as a selectable marker, and if possible inactivate the ampRgene. As we do not wish to alter the properties of pTETnir15, which makeit a very effective expression system in live vaccines, we wished toincorporate the minimum number of changes in the plasmid.

The ampicillin resistance gene was removed and replaced with theherbicide resistance gene by the following strategy. The 3727 bppTETnir15 was digested with the restriction enzymes Asp 700 and Pst1which cut exclusively within the 860 bp ampR gene to release a 354 bpfragment located towards the 5'-end of the gene. The 3373 bp remnantvector was gel-purified and was now ready for cloning in the bar gene.

The plasmid pSCB-1 contains the bar gene and was obtained from PBICambridge. A bar gene expression cassette was synthesised by thepolymerase chain reaction (PCR; Saiki et al 1988) from pSCB-1.

TATGAATCAGTTCCATCT ACCATGAGCCCAGAACGA(SEQ ID NO:1)

TATCTGCAGTTAGATCTC GGTGACGGGCA(SEQ ID NO:2)

The reaction was performed using sense and antisense primers designed toamplify the complete open reading frame of the bar gene. In addition tofacilitate the cloning of the bar gene the sense primer was tailoredwith the recognition sequence of the restriction enzyme Asp700 and theantisense primer was tailored with the recognition sequence for Pst1.The product was gel-purified and digested with Asp700 and Pst1,resulting in a bar gene cassette of approximately 579 bp, and thencloned into the residual 3373 bp pTETnir15 plasmid which had also beencut with the respective enzymes. The resulting plasmid was designatedpBAT1.

This generated an open reading frame in pBAT1 composed of 59 codons fromthe ampR gene followed by all the codons from the bar gene cassetteterminating in a stop codon, followed by the remaining 92 codons fromthe ampR gene. This results in the expression of a short 59 amino acidN-terminal fusion of AmpR to the full length Bar. This approach has theadvantage that it allows the expression of the bar gene by the naturalampR promoter, retains the integrity of the ribosome binding sequence,and allows the bar gene to utilise the signal sequence of ampR.Furthermore, this strategy allows the ampR gene to be partially deletedand insertionally inactivated.

Experiments to Test the Ability of the Bar Gene in PBATI to ConferResistance to PPT in Salmonella

The construct was electroprated into electrocompetent C5htrA cells andtransformants selected by adding cells to molten (48° C.) minimal agarsupplemented with M9 salts. Transformed C5htrA cells harbouring pBAT1were selected by the addition of PPT (250 ug/ml) and 100 ul dropletsspotted onto a petridish. The plates were then incubated at 37° C. for48 hours.

The agar droplets were then transferred to minimal broth mediasupplemented with M9 salts containing 375 ug/ml of PPT. The cells weregrown shaking at 37° C. overnight. A stock of the culture was made priorto harvesting the cells and purifying the pBAT1 plasmid DNA. Theidentity of the construct was verified by restriction enzyme mappingwith EcoR1 and Pst1.

Furthermore pBAT1 (C5htrA) is inhibited from growth in mediasupplemented with ampicillin (50 ug/ml), suggesting there is noremaining residual activity from the partially deleted and insertionallydisrupted ampR gene as expected.

In summary pBAT1 has been constructed. This plasmid expresses the bargene and is capable of conferring PPT resistance to the host S.typhimurium C5htrA vaccine strain allowing this herbicide to be used asa selective marker for cells harbouring this construct.

Experiments to Show that the Bar Gene does not Alter the Physiology ofthe Host Salmonella

For the bar gene to be a selective marker of practical value in avaccine, host vaccine cells harbouring the constructs containing themarker should retain their original properties. For example the plasmidshould be able to continue to express guest antigens, and remain stableby not being segregated and lost from the host cell population in theabsence of marker selection.

The ability of pBAT1 (C5htrA) to express TetC was compared to pTETnir15(C5HtrA) by SDS-PAGE and immunoblotting of these cells. In each caseprobing the immunoblots with a rabbit anti TetC polyclonal sera revealedno difference in the expression levels and stability of the 50 kDa TetCband.

Thus it can be concluded that there is no aberrant expression of TetCfrom the pBAT1 construct.

The ability of pBAT1 to be stably retained in C5HtrA in the absence ofmarker selection was investigated and compared to pTETnir15 in C5HtrA.

The strains were grown in the liquid media already described abovesupplemented with either PPT (375 ug/ml) or ampicillin (50 ug/ml)shaking overnight at 37° C. The following day the cultures were diluted1 in a 100 into fresh media, each with and without the respectiveselective marker. The four cultures were again shaken at 37° C. and thefollowing culture dilutions ranging 10⁶ to 10⁸ plated out onto minimalagar plates, again with and without the selective marker for each of thefour cultures.

The number of colonies arising from each of the four cultures plated onminimal media with and without selection was determined. For pBAT1(C5HtrA) and pTETnir15 (C5HtrA) the number of colonies counted fromliquid cultures which had been grown in the presence and absence ofselection yielded the same number of colonies on plates with/without theselective marker. This would strongly suggest the plasmid pBAT1 is asstably inherited as pTETnir15 in the absence of selection with eitherPPt or ampicillin respectively.

Thus it can be concluded that the bar gene product does not place thehost cells harbouring the construct at a selective disadvantage. Thisimplies that the expression of the bar gene does not significantly alterthe physiology of the host cell and this of course is a highly desirableproperty.

Salmonella typhi Experiments to Show that Salmonella typhi is Sensitiveto PPT

To investigate the possibility that S.typhi is sensitive to thebacteriocidal effects of PPT, the following experiment was conducted.The S.typhi stain 541Ty [Edwards and Stocker, 1981] was selected as amodel strain. 541Ty has a number of nutritional requirements and wastherefore grown in minimal media with M9 salts and the followingsupplements. To 5 ml of media 25 ul of each of the following were added:1% L-serine, 1% pyridoxine, 0.1% 2,3-dihydroxybenzoic acid (DHB), 1%L-histidine, 1% L-adenine, 1% cystine, 1% L-tryptophan, and Aro mix (1%L-phenylalanine, 1% L-tyrosine, 1% L-tryptophan, 0.1% para-aminobenzoicacid (PABA), 0.1% DHB).

The strain 541Ty was incubated overnight at 37° C. under normal cultureconditions in minimal media with the 541Ty supplements described aboveeither alone, and also with PPT at 350 ug/ml. After the incubation itwas observed that the culture supplemented with PPT, in contrast to theculture lacking PPT, had failed to grow.

This experiment therefore established that our selected S.typhi hoststrain is indeed susceptible to the bacteriocidal effects of PPT.

REFS:

Construction of ΔaroA his Δpur strians of Salmonella typhi.

Edwards F., Stocker B.

Journal of Bacteriology 170: 3991-3995, 1988.

Experiments to Test the Ability of the Bar Gene in pBAT1 to ConferResistance to PPT in Salmonella typhi

The construct pBAT1 was electroplated into electrocompetent 541Ty cellsand transfornants selected by adding cells to molten (48° C.) minimalagar with the supplements already described above, and also PPT (350ug/ml) to select for transformants. The plates were then incubated at37° C. for 48 hours.

The recombinant clones were picked and grown shaking for 36 hours inminimal media with the 541Ty supplements already described, and PPT (350ug/ml). Stocks of the clones were made prior to inoculating thecultures. Cells were harvested and the plasmid pBAT1 isolated. Theidentity of the construct was verified by restriction enzyme mappingwith Eco R1and Pstl.

In summary, the plasmid pBAT1 expresses the bar gene and is capable ofconferring PPT resistance to the host S.typhi strain, allowing thisherbicide to be used as a selective marker for S.typhi cells harbouringthis construct.

Experiments to Show that the Bar Gene does not Alter the Physiology ofthe Host Salmonella typhi

For the bar gene to be a selective marker of practical value in avaccine, host vaccine cells harbouring the constructs containing themarker should retain their original properties. For example the plasmnidshould be able to continue to express guest antigens, and remain stableby not being segregated and lost from the host cell population in theabsence of marker selection.

The ability of pBAT 1(541Ty) to express TetC was compared topTETnir15(541Ty) by SDS-PAGE and immunoblotting of these cells. In eachcase probing the immunblots with a rabbit anit TetC polyclonal serarevealed no difference in the expression levels and stability of the 50kDa TetC band.

Thus it can be concluded that there is no aberrant expression of TetCfrom the pBAT1 construct.

The ability of pBAT1 to be stably retained in 541Ty in the absence ofmarker selection was investigated and compared to pTETnir15 in 541Ty.

The strains were grown in the liquid media already described abovesupplemented with either PPT (375 ug/ml) or ampicillin (50 ug/ml)shaking overnight at 37° C. The following day the cultures were diluted1 in a 100 into fresh media, each with and without the respectiveselective marker. The four cultures were again shaken at 37° C. and thefollowing culture dilutions ranging 10⁶ to 10⁸ plated out onto minimalagar plates with the 541Ty supplements, again with and without theselective marker for each of the four cultures.

The number of colonies arising from each of the four cultures plated on541Ty minimal media with and without selection was determined. For pBAT1(541Ty) and pTETnir15 (541Ty) the number of colonies counted from liquidcultures which had been grown in the presence and absence of selectionyielded the same number of colonies on plates with/without the selectivemarker. This would strongly suggest the plasmid pBAT1 is as stablyinherited as pTETnir15 in the absence of selection with either PPT oramnpicillin respectively.

Thus it can be concluded that the bar gene product does not place thehost S.typhi cells harbouring the construct at a selective disadvantage.This implies that the expression of the bar gene does not significantlyalter the physiology of the host S.typhi and this of course is a highlydesirable property.

SUMMARY

We have therefore shown that it is possible to produce a vaccine for usein animals, and in particular for use in humans, which maybe eitherunivalent or multivalent, but in any event, comprises a transformed hostcell wherein successful transformation is determined having regard tothe resistance of the host cell to a pre-selected substance by virtue ofthe transformation of the said host cell with a gene conferringresistance to said substance, other than an antibiotic resistance gene.

REFERENCES

Gonzalez C., Hone D., Noriega F. R., Tacket C. O., Davis J. R., LosonskyG., Nataro J. P., Hoffman S., Malik A., Nardin E., Sztein B., Heppner D.G., Fouts T. R., Isibasi A., L-evine M. M. Journal of InfectiousDiseases 169: 927-31, 1994.

Murakami T., Anzai H., Imai S., Satoh H., Nagaoka K., Thompson C. J.Molecular and General Genetics, 205: 42-50, 1986.

Thompson C. J., Morva N. R., Tizard R., Crameri R., Davis J. E.,Lauwereys M., Botterman J. EMBO Journal, 6: 2519-2523, 1987.

Khan C. M. A., Villarreal-Ramos B., Pierce R. J., Riveau G., Demarco deHormaeche, McNeill H., Ali T., Fairweather N., Chatfield S., Capron A.,Dougan G., Hormaeche C. E. Proceedings of the National Academy ofSciences, USA. 91: 11261-11265, November 1994.

Chabalgoity J. A., Khan C. M. A., Nash A. A., Horrnaehe C. E. MolecularMicrobiology 19: 791-801, 1996.

Chatfield S. N., Charles I. G., Makoff A. J., Oxer M. D., Dougan G.,Slater D., Fairweather N. F., Bio/Technology 10: 888-892, 1992.

Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., HornG. T., Mullis K. B., Ehrlich H. A. Science 239: 487-491, 1988.

D'Halluin K., De Block M., Denecke J., Janssens J., Leemans J.,Reynaerts A., Botterman J., Methods in Enzymology 216: 415-426, 1992.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 2                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 36                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: syntheticION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 1                                                           #         36aatcag ttccatctac catgagccca gaacga - #                           - <210> SEQ ID NO 2                                                           <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: SyntheticION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 2                                                           #              29  tagatctcgg tgacgggca                                       __________________________________________________________________________

What is claimed is:
 1. A transformed cell which has been engineered soas to express at least one antigen and at least one safe, selectablemarker which confers on said cell resistance to an agent, other than anantibiotic, which would otherwise deleteriously affect the growth ofsaid cell, wherein said cell is an attenuated strain of its naturalcounterpart.
 2. A transformed cell according to claim 1 wherein saidcell has been engineered to express a plurality of antigens.
 3. Atransformed cell according to claim 1 wherein multiple copies of thegene encoding said at least one antigen are provided.
 4. A transformedcell according to claim 1 wherein said at least one antigen isoperatively linked to a high expression promoter.
 5. A transformed cellaccording to claim 4 wherein said promoter is inducible.
 6. Atransformed cell according to claim 5 wherein said promoter is induciblein vivo.
 7. A transformed cell according to claim 1 wherein said cell isadapted to express at least a fragment of tetanus toxoid.
 8. Atransformed cell according to claim 7 wherein said fragment is thehighly immunogenic but atoxic fragment C (TetC) from tetanus toxin.
 9. Atransformed cell according to claim 1 wherein said cell is engineered toexpress an agent that enhances the immunogenicity of the cell.
 10. Atransformed cell according to claim 1 wherein said safe, selectablemarker is a product of the bar gene.
 11. A vector comprising at leastone gene encoding an agent, other than an antibiotic resistance agent,that counters, or advantageously affects, the otherwise deleteriouseffects of a substance to which a cell that is to be transformed by saidvector is susceptible; and at least one gene encoding an antigen from atleast one pathogen known to cause disease in animals.
 12. A vectoraccording to claim 11 wherein said vector comprises at least one copy ofthe bar gene.
 13. A vector according to claim 11 wherein said vectorincludes multiple copies of said gene encoding said antigen.
 14. Avector according to claim 11 wherein said vector includes genes encodinga plurality of different antigens all selected from pathogens which arecapable of causing disease in animals.
 15. A vector according to claim11 wherein said at least one gene encoding said antigen is operativelylinked to a high expression promoter.
 16. A vector according to claim 15wherein said promoter is inducible.
 17. A vector according to claim 16wherein said promoter is inducible in vivo.
 18. A vector according toclaim 11 wherein said vector includes an antibiotic resistance genewhich is made insertionally inactive by the insertion therein of atleast one gene encoding a product that, directly or indirectly, confersresistance to a substance, other than an antibiotic, which substance iscapable of deleteriously affecting a cell to be transformed by saidvector.
 19. A vector according to claim 18 wherein said gene encodingresistance to said substance is the bar gene.
 20. A vector according toclaim 18 wherein, optionally or additionally, a gene encoding at leastone antigen is inserted into said antibiotic resistance gene so as torender said gene insertionally inactive.
 21. A transformed cellaccording to claim 1 wherein the cell is a Salmonella cell.
 22. Atransformed cell according to claim 21 wherein the cell is a Salmonellatyphi cell.